BrdU-CLIP

CLIP with BrdU Affinity Purification

BrdU-CLIP sequences the binding sites of RBPs with single-nucleotide resolution. BrdU-CLIP fixes a major problem in HITS-CLIP where 5′ adapters are not attached to the cDNA due to premature termination of reverse transcription (Weyn-Vanhentenryck et al., 2014). BrdU-CLIP uses BrdUTPs and primers with 3′ and 5′ adapters during reverse transcription. This method enables greater cDNA yield and attaches both 5′ and 3′ adapters in a single step.

First, RNA-protein complexes are UV-crosslinked, digested with RNase, and immunoprecipitated. 3′ adapters are ligated to the RNA and reverse-crosslinked by proteinase K digestion. Some peptides from the crosslink remain bonded with the RNA strands, even after reverse-crosslinkage. RT is carried out using BrdUTPs and primers containing 3′ and 5′ adapters separated by an apurinic/apyrimidinic endonuclease (APE) cleavage site. The resultant cDNA is purified, circularized, and further purified by BrdU pull-down. Next, the cDNA is linearized by cleaving at the APE site, PCR-amplified, and sequenced.

Advantages:

  • Sequences RNA-binding sites of RBPs with single-nucleotide resolution
  • Captures both truncated and nontruncated cDNAs, unlike HITS-CLIP
  • Provides higher cDNA yield due to BrdUTP
  • Attaches 3′ and 5′ adapters in one RT step

Disadvantages:

  • Tube-column transfers can result in major loss of material (Zarnegar et al., 2016)
  • Not yet adopted widely by the scientific community


Reagents:

Illumina Library prep and Array Kit Selector



Reviews:

Scotti M. M. and Swanson M. S. RNA mis-splicing in disease. Nat Rev Genet. 2016;17:19-32

Vuong C. K., Black D. L. and Zheng S. The neurogenetics of alternative splicing. Nat Rev Neurosci. 2016;17:265-281

Hsiao Y.-H. E., Cass A. A., Bahn J. H., Lin X. and Xiao X. Global Approaches to Alternative Splicing and Its Regulation√£Recent Advances and Open Questions. Transcriptomics and Gene Regulation. 2016;37-71

Bangru S. and Kalsotra A. Advances in analyzing RNA diversity in eukaryotic transcriptomes: peering through the Omics lens. F1000Research. 2016;5:2668

Flynn R. A., Martin L., Spitale R. C., Do B. T., Sagan S. M., et al. Dissecting noncoding and pathogen RNA-protein interactomes. RNA. 2015;21:135-143



References:

Weyn-Vanhentenryck S. M., Mele A., Yan Q., et al. HITS-CLIP and integrative modeling define the Rbfox splicing-regulatory network linked to brain development and autism. Cell Rep. 2014;6:1139-1152